We previously reported that pharmacological preconditioning of rabbit hearts with
acetylcholine involves activation of
phosphatidylinositol 3-kinase (PI3-K) through transactivation of the
epidermal growth factor receptor (EGFR). Transactivation is thought to be initiated by cleavage of membrane-bound pro-
heparin-binding EGF-like growth factor (
HB-EGF) by a membrane
metalloproteinase thus releasing
HB-EGF which binds to the EGFR. This pathway leads to redox signaling with the generation of
reactive oxygen species (ROS) by mitochondria. We tested whether preconditioning's physiological triggers,
bradykinin and
opioid, also signal through the EGFR. Both
bradykinin and the synthetic delta-
opioid agonist
DADLE increased ROS production in isolated cardiomyocytes by approximately 50%.
DADLE's effect was abrogated by either
metalloproteinase inhibitor III (MPI) or the
diphtheria toxin mutant
CRM-197 which blocks
heparin-binding
EGF shedding indicating that
DADLE signals through EGFR transactivation. MPI also blocked
DADLE's
infarct-sparing effect in whole hearts. Additionally, blocking
Src kinase (a component of the EGFR's signaling complex) with PP2 or PI3-K with
wortmannin blocked
DADLE's effect on cardiomyocyte ROS production and PP2 blocked
DADLE's salvage of ischemic myocardium. Finally,
DADLE increased phosphorylation of Akt and extracellular signal-regulated
protein kinases (ERK) 1/2 in left ventricular myocardium, and this increase was blocked by the EGFR antagonist
AG1478. On the other hand, neither MPI nor
CRM-197 prevented
bradykinin from increasing ROS production, and MPI did not affect
bradykinin's
infarct-sparing effect in intact hearts. Conversely, both PP2 and
wortmannin blocked
bradykinin's effect on ROS generation and also aborted
bradykinin's cardioprotective effect in intact hearts. While
bradykinin also increased phosphorylation of Akt and ERK in myocardium, that increase was not affected by
AG1478. Hence
bradykinin, unlike
acetylcholine or
opioid, does not transactivate EGFR, although all 3 agonists do signal through Src and PI3-K.